5 research outputs found
Development of a Fluorescence Internal Quenching Correction Factor to Correct Botulinum Neurotoxin Type A Endopeptidase Kinetics Using SNAPtide
No full textBotulinum neurotoxins (BoNTs), which are highly toxic
proteins responsible for botulism, are produced by different strains
of Clostridium botulinum. These various strains of
bacteria produce seven distinct serotypes, labeled A–G. Once
inside cells, the zinc-dependent proteolytic light chain (LC) degrades
specific proteins involved in acetylcholine release at neuromuscular
junctions causing flaccid paralysis, specifically synaptosomal-associated
protein 25 (SNAP-25) for botulinum neurotoxin type A (BoNT/A). BoNT
endopeptidase assays using short substrate homologues have been widely
used and developed because of their ease of synthesis, detection limits,
and cost. SNAPtide, a 13-amino acid fluorescence resonance energy
transfer (FRET) peptide, was used in this study as a SNAP-25 homologue
for the endopeptidase kinetics study of BoNT/A LC. SNAPtide uses a
fluorescein isothiocyanate/4-((4-(dimethylamino)phenyl)azo) benzoic
acid (FITC/DABCYL) FRET pair to produce a signal upon substrate cleavage.
Signal quenching can become an issue after cleavage since quencher
molecules can quench cleaved fluorophore molecules in close proximity,
reducing the apparent signal. This reduction in apparent signal provides
an inherent error as SNAPtide concentrations are increased. In this
study, fluorescence internal quenching (FIQ) correction factors were
derived using an unquenched SNAPtide peptide to quantify the signal
quenching over a range of SNAPtide concentrations and temperatures.
The BoNT/A LC endopeptidase kinetics at the optimally active temperature
(37 °C) using SNAPtide were studied and used to demonstrate the
FIQ correction factors in this study. The FIQ correction factors developed
provide a convenient method to allow for improved accuracy in determining
and comparing BoNT/A LC activity and kinetics using SNAPtide over
a broad range of concentrations and temperatures
Evolutionary Features in the Structure and Function of Bacterial Toxins
No full textToxins can function both as a harmful and therapeutic molecule, depending on their concentrations. The diversity in their function allows us to ask some very pertinent questions related to their origin and roles: (a) What makes them such effective molecules? (b) Are there evolutionary features encoded within the structures of the toxins for their function? (c) Is structural hierarchy in the toxins important for maintaining their structure and function? (d) Do protein dynamics play a role in the function of toxins? and (e) Do the evolutionary connections to these unique features and functions provide the fundamental points in driving evolution? In light of the growing evidence in structural biology, it would be appropriate to suggest that protein dynamics and flexibility play a much bigger role in the function of the toxin than the structure itself. Discovery of IDPs (intrinsically disorder proteins), multifunctionality, and the concept of native aggregation are shaking the paradigm of the requirement of a fixed three-dimensional structure for the protein’s function. Growing evidence supporting the above concepts allow us to redesign the structure-function aspects of the protein molecules. An evolutionary model is necessary and needs to be developed to study these important aspects. The criteria for a well-defined model would be: (a) diversity in structure and function, (b) unique functionality, and (c) must belong to a family to define the evolutionary relationships. All these characteristics are largely fulfilled by bacterial toxins. Bacterial toxins are diverse and widely distributed in all three forms of life (Bacteria, Archaea and Eukaryotes). Some of the unique characteristics include structural folding, sequence and functional combination of domains, targeting a cellular process to execute their function, and most importantly their flexibility and dynamics. In this work, we summarize certain unique aspects of bacterial toxins, including role of structure in defining toxin function, uniqueness in their enzymatic function, and interaction with their substrates and other proteins. Finally, we have discussed the evolutionary aspects of toxins in detail, which will help us rethink the current evolutionary theories. A careful study, and appropriate interpretations, will provide answers to several questions related to the structure-function relationship of proteins, in general. Additionally, this will also allow us to refine the current evolution theories
A novel role of C-terminus in introducing a functionally flexible structure critical for the biological activity of botulinum neurotoxin
No full textAbstractBotulinum neurotoxin (BoNT) is responsible for botulism, a clinical condition resulting in flaccid muscle paralysis and potentially death. The light chain is responsible for its intracellular toxicity through its endopeptidase activity. Available crystal structures of BoNT/A light chains (LCA) are based on various truncated versions (tLCA) of the full-length LCA (fLCA) and do not necessarily reflect the true structure of LCA in solution. The understanding of the mechanism of action, longevity of intoxication, and an improved development of endopeptidase inhibitors are dependent on first having a better insight into the structure of LCA in solution. Using an array of biophysical techniques, we report that the fLCA structure is significantly more flexible than tLCA in solution, which may be responsible for its dramatically higher enzymatic activity. This seems to be achieved by a much stronger, more rapid binding to substrate (SNAP-25) of the fLCA compared to tLCA. These results suggest that the C-terminus of LCA plays a critical role in introducing a flexible structure, which is essential for its biological function. This is the first report of such a massive structural role of the C-terminus of a protein being critical for maintaining a functional state.</jats:p
Diseño latinoamericano: diez miradas a una historia en construcción
No full textEn los últimos veinte años, las publicaciones sobre los diseños en América Latina han comenzado a revelar la diversidad y riqueza que presentan sus distintas historias. Una diversidad que no responde solamente a las características disímiles que asumen las disciplinas proyectuales en los distintos países de la región, sino también a las diferentes concepciones, ideas y enfoques con los que se ha construido y configurado el objeto diseño a lo largo de la historia de cada país.
Este libro ha querido poner de relieve ese cruce de caminos, interrogar ese lugar pleno de diversidades. Como resultado de un proceso consciente, se ofrecen diez ensayos escritos por autores provenientes de las instituciones universitarias más destacadas de la región que abordan, en primera instancia, la historiografía del diseño —en un sentido amplio— en México, Venezuela, Colombia, Chile, Brasil y Argentina.
El lector encontrará en los textos de Marina Garone Gravier, Dina Comisarenco Mirkin, Alberto Sato, Marisol Orozco Álvarez, Juan Camilo Buitrago Trujillo, Pedro Álvarez Caselli y Alejandra Neira Román, Ana Utsch y Bruno Guimarães Martins, Marcos da Costa Braga, Verónica Devalle, Horacio Caride Bartrons y Alejo García de la Cárcova no solamente perspectivas fundamentadas y explicaciones documentadas, sino que contará con una obra pertinente y actual para una disciplina en construcción
